The regimented and conserved pattern of the vascular network suggests that specific genetic programs coordinate its formation. Both the nervous and vascular systems begin as central axial structures that send sprouts to distal targets and form complex, highly reproducible and often superimposable networks. The development of the neural network is regulated by combinations of attractive and repulsive cues that guide growth cones. Secreted Slit proteins provide repulsive signals through their cognate Robo receptors and are critical for regulating whether axons cross the midline. Recently, we found that the vascular endothelium exclusively expresses a novel member of the Robo receptor family, Robo4. Slit-Robo4 signaling blocks the migration of cells to fibroblast growth factors and vascular endothelial growth factor. Here, we propose to understand the role of Robo signaling in vascular guidance. In Specific Aim 1, we will investigate the role of Robo4 signaling in endothelial sprouting. Using cell biology assays, we will determine whether the function of Slit-Robo4 signaling in endothelial cell biology is analogous to the role of Slit-Robo signaling in axonal guidance. We predict that Slit-Robo4 signaling will disrupt endothelial migration, sprouting, adhesion and cytoskeletal architecture but have no effect on proliferation or tube formation. Specific Aim 2 will examine the molecular mechanism of Robo4 signaling. Using yeast-two hybrid analyses and immunoprecipitations, we will identify intracellular proteins that bind to Robo4 and mediate its downstream signaling cascade. Our studies have revealed a potential link between Robo4 receptors and the paxillin family of proteins. We will define the biochemical interactions between Robo4 and downstream proteins, and the functional roles of these interactions. In Specific Aim 3, we will examine the role of Robo4 in vascular development using knockout mice. Preliminary data suggest that ablating Robo4 results in increased vascular sprouting around and within the neural tube. Together, the studies outlined in this grant application seek to demonstrate that nerves and vessels exploit a common signaling mechanism, the Slit-Robo pathway, for guiding the formation of their respective networks along specific tracts. [unreadable] [unreadable]